Project Summary. Early life stress (ELS) profoundly and negatively affects mental health into adulthood, disrupting neural circuits for threat estimation. This proposal is designed to test the overarching hypothesis that ELS reprograms signaling of DR gamma-aminobutyric acid (GABA), serotonin (5HT), and non-GABA-non-5HT neurons to inflate BLA threat estimates and exaggerate fear. Specifically, DR 5HT neurons and GABA neurons are responsive to threatening cues, while non-GABA-non-5HT neurons are responsive to surprising omission of aversive outcomes, termed a negative prediction error (?PE). ELS reprograms DR by diminishing GABA inhibition of 5HT, while concurrently diminishing non-GABA-non-5HT output. The net result of ELS on DR function is to increase 5HT output to inflate BLA threat estimation and exaggerate fear. We have designed an ELS procedure in which pre-pubertal rats experience multiple categories of physical stressors. In adulthood, accurate threat estimation is assessed with a fear discrimination paradigm in which three cues signal different probabilities of foot shock: danger (p=1.00), uncertainty (p=0.25), and safety (p=0.00). In Aim 1, we will record single-unit activity from the DR and BLA during fear discrimination in normal or ELS rats with DR intact, DR- 5HT neuron deletion or DR-GABA neuron deletion. Neuron-type deletion will be achieved by using specific transgenic rat lines (GAD-cre or TPH2-cre) in combination with cre-dependent caspases. Primary analyses will focus on neural activity during the cue period, when threat estimates are generated, and post-cue period, when ?PEs are generated and will directly determine the impact of ELS, and neuron-type deletion, on single-unit activity. In Aim 2, we will use optogenetic inhibition during the cue period, when threat estimates are generated, and the post-cue period, when ?PEs are generated, to demonstrate causal roles for, DR GABA, DR 5HT, DR Non-5HT populations, and DR-to-BLA projections in fear to uncertainty. This will uncover causal roles for DR neuron-types, and direct DR to BLA projections, in accurate threat estimation in normal and ELS individuals. In Aim 3, we will combine neuron-type-specific optogenetic excitation of the DR with single-unit recording in the BLA of ELS rats. DR GABA neurons will be stimulated during cue periods, and DR Non-5HT neurons will be stimulated during ?PE periods, in order to restore appropriate fear, and BLA neural activity, to uncertainty. This proposal will demonstrate that ELS reprograms neuron-type-specific signaling in the DR-to- BLA circuit to inflate threat estimation. The results will uncover novel targets for pharmacotherapies to restore accurate estimation in ELS individuals. The long term goal of this research is to map a complete neural for accurate threat estimation, reveal how ELS disrupts circuit function, and use this knowledge to design fully effective therapies to restore accurate threat estimation in ELS-affected individuals.